The inhibition of the enzyme adenylate cyclase by hormone receptors has many important physiological consequences. While the mechanism of the inhibition is unknown, it is likely that a guanine nucleotide binding component couples the receptor with adenylate cyclase. The overall objective of the proposed research is to investigate the mechanism of the alpha2 adrenergic receptor-mediated inhibition of adenylate cyclase in platelets. The problem will be approached from two viewpoints: (I) characterization of the alpha2 receptor and (II) examination of the interaction of the inhibitory regulatory components with adenylate cyclase. In (I) the alpha2 receptors will be solubilized from platelet plasma membranes with the detergent digitonin and identified with (3H) yohimbine subsequent to solubilization. The receptor will be partially purified initially using traditional techniques. Since the platelet's alpha2 receptor is a glycoprotein, lectin chromatography will be used to purify the receptor. Also, a number of affinity gels involving immobilized alpha adrenergic antagonists will be evaluated. Gel permeation HPLC may be required for the definitive purification. Either membrane-bound or partially purified solubilized alpha2 receptor will be used to raise monoclonal antibodies to the receptor; antibodies to the receptor will be detected by immunoprecipitation. The antibodies will be used to expedite receptor purification and to probe its structural relationship to other catecholamine receptors. (II) Preliminary experiments will be undertaken to determine directly whether Mn++ perturbs the interaction between the stimulatory regulatory component from pigeon erythrocytes (labeled with 32P by cholera toxin) and adenylate cyclase. If Mn++ does prevent this interaction, it will be used as an additional probe to discern the interaction of the inhibitory regulatory component with adenylate cyclase on sucrose density gradients. Reconstitution experiments will be designed to detect and possibly separate the inhibitory and stimulatory regulatory components of adenylate cyclase.